Abstract

The transcriptional complex hypoxia-inducible factor-1 (HIF-1) has
emerged as an important mediator of gene expression patterns in tumors,
although the range of responding genes is still incompletely defined.
Here we show that the tumor-associated carbonic anhydrases (CAs) are
tightly regulated by this system. Both CA9 and
CA12 were strongly induced by hypoxia in a range of
tumor cell lines. In renal carcinoma cells that are defective for the
von Hippel-Lindau (VHL) tumor suppressor, up-regulation of these CAs is
associated with loss of regulation by hypoxia, consistent with the
critical function of pVHL in the regulation of HIF-1. Further studies
of CA9 defined a HIF-1-dependent hypoxia response
element in the minimal promoter and demonstrated that tight regulation
by the HIF/pVHL system was reflected in the pattern of CA IX expression
within tumors. Generalized up-regulation of CA IX in VHL-associated
renal cell carcinoma contrasted with focal perinecrotic expression in a
variety of non-VHL-associated tumors. In comparison with vascular
endothelial growth factor mRNA, expression of CA IX demonstrated a
similar, although more tightly circumscribed, pattern of expression
around regions of necrosis and showed substantial although incomplete
overlap with activation of the hypoxia marker pimonidazole. These
studies define a new class of HIF-1-responsive gene, the activation of
which has implications for the understanding of hypoxic tumor
metabolism and which may provide endogenous markers for tumor hypoxia.

INTRODUCTION

Tumor hypoxia is an important indicator of cancer prognosis; it is
associated with aggressive growth, metastasis, and poor response to
treatment
(1, 2)
. Of potential importance for
understanding these effects is the role of hypoxia in regulating
patterns of gene expression
(3,
4,
5,
6)
. Studies of gene
expression have defined several classes of hypoxia-inducible genes that
are up-regulated in hypoxic regions of tumors and demonstrated that
activation of the transcriptional complex
HIF-1
3
is a key mediator of many of these effects
(7,
8,
9)
.

Genes that are up-regulated by microenvironmental hypoxia through
activation of HIF include glucose transporters, glycolytic enzymes, and
angiogenic growth factors
(5, 10, 11)
. For some HIF
targets such as VEGF, a clear function in promoting tumor growth is
established
(12)
. However, the full range of HIF target
genes has not yet been defined, and identification of additional genes
responding to this pathway is likely to provide further insights into
the consequences of tumor hypoxia and HIF activation. Indirect support
for the importance of microenvironmental activation of HIF has also
been provided by recent demonstrations of constitutive activation of
HIF after inactivation of the VHL tumor suppressor gene
(13)
and amplification of the HIF response by other
oncogenic mutations
(14,
15,
16,
17)
. Mutations in VHL cause the
familial syndrome and are also found in the majority of sporadic RCCs
(18)
. The gene product pVHL forms part of a
ubiquitin-ligase complex
(19, 20)
that targets HIF-α
subunits for oxygen-dependent proteolysis
(13, 21)
. In
VHL-defective cells, HIF-α is stabilized constitutively, resulting in
up-regulation of hypoxia-inducible genes such as VEGF
(13)
. Although the pVHL ubiquitin-ligase complex may have
other targets
(20)
and other functions of pVHL have been
proposed that may contribute to tumor suppressor effects
(22, 23)
, these recent findings raise important questions as to the
range of genes affected by constitutive HIF activation and the role of
these genes in oncogenesis.

In this respect, one interesting group of genes is the tumor-associated
transmembrane CAs CA9(24,
25,
26,
27)
and
CA12(28, 29)
. CAs catalyze the reversible
hydration of carbon dioxide to carbonic acid
(30)
,
providing a potential link between metabolism and pH regulation. The
membrane-linked isoforms CA9 and CA12 were
identified by RNA differential display as genes that are down-regulated
by pVHL
(29)
, although the effect of hypoxia was not
examined and the mechanism of regulation was not defined.
Interestingly, CA9 can confer a variety of features of the
transformed phenotype when transfected into NIH 3T3 cells
(24)
.

In this study, we demonstrate that in contrast to constitutive
up-regulation in pVHL-defective cell lines, both CA9 and
CA12 are strongly induced by hypoxia in a broad range of
other cell types. The induction of CA9 by hypoxia was
striking and has been studied in detail. We show that the
CA9 promoter is tightly regulated by a HIF-responsive HRE
close to its transcriptional start site, and that the gene product is
expressed in a perinecrotic manner in many types of human cancer,
overlapping with VEGF mRNA and the hypoxia marker pimonidazole. In
keeping with constitutive activation of HIF after inactivation of pVHL,
the focal pattern of expression observed in most tumors contrasted with
that observed in RCCs, where CA IX was globally up-regulated. Our
findings define a new biochemical pathway that is regulated by HIF,
suggest that CA IX may be a useful marker for HIF activation either by
microenvironmental hypoxia or genetic events such as VHL inactivation,
and provide additional insights into mechanisms by which the HIF
pathway might mediate effects on tumor metabolism.

RNA Analysis.

Total RNA was extracted by a modified acid/guanidinium
thiocyanate/phenol/chloroform method (RNAzol B; Cinna/Biotec
Laboratories), dissolved in hybridization buffer (80% formamide, 40
mm PIPES, 400 mm sodium chloride, and 1
mm EDTA, pH 8) and analyzed by RPA. To generate appropriate
riboprobe templates, cDNA fragments of human CA9
(nucleotides 3632–3771, accession number Z54349) and CA12
(nucleotides 301–450, accession number AF037335) were amplified by PCR
and ligated into pSP72 (Promega). DNA templates for generating
32P-labeled RNA probes were linearized for
16 h with BglII and transcribed with SP6 RNA
polymerase. For CA9 and CA12, RPAs were performed
on 30 μg of total RNA, using an internal control assay for U6 small
nuclear RNA as described
(13)
.

Construction of Reporter Plasmids.

To generate plasmids p-506 and p-173, sequences of the CA9
gene between −506 and +43 relative to the transcriptional start site
were amplified by PCR from genomic DNA. PCR products were ligated into
pGL3-basic, a promoterless and enhancerless luciferase expression
vector (Promega). To generate plasmids p-36, MUT1, and MUT2,
complementary oligonucleotides with ends corresponding to the 5′
restriction cleavage overhangs of BglII and MluI
were annealed and ligated into
BglII/MluI-digested pGL3-basic. Oligonucleotides
(sense strand) were: p-36 (forward),
5′-cgcgCTCCCCCACCCAGCTCTCGTTTCCAATGCACGTACAGCCCGTACACACCG-3′;
MUT1 (forward),
5′-cgcgCTCCCCCACCCAGCTCTCGTTTCCAATGCTTTTACAGCCCGTACACACCG-3′;
MUT2 (forward),
5′-cgcgCTCCCCCACCCAGCTCTCGTTTCCAATGCAAGTACAGCCCGTACACACCG-3′.
Nucleotides introduced for cloning are lowercase; mutations are
underlined. All CA9 promoter sequences were confirmed by
dideoxy sequence analysis.

Transient Expression Assays.

Cells at ∼70% confluence in 60-mm dishes were transfected with 1μ
g of a luciferase reporter construct and 0.4 μg of control
plasmid, pCMV-βgal (Promega), using FuGENE 6 (Roche Diagnostic)
according to the manufacturer’s instructions. Cells were then
incubated at 20% O2 for 8 h, followed by
20% or 0.1% O2 for 16 h.

Luciferase activity was determined in cell lysates using a commercial
assay system (Promega) and a TD-20e luminometer (Turner Designs).β
gal activity in cell lysates was measured using
ο-nitrophenyl-β-d-galactopyranoside
as substrate in a 0.1 m phosphate buffer (pH 7.0)
containing 10 mm KCl, 1 mm
MgSO4, and 30 mmβ
-mercaptoethanol. To correct for variable transfection efficiencies
between experimental conditions, the luciferase:βgal ratio was
determined for each sample. For cotransfection assays, cells also
received 0.1–1 μg each of pCDNA3/HIF-1α or pCDNA3/HIF-2α
containing the entire human HIF-1α or HIF-2α open reading frame,
respectively. Transfections were balanced with various amounts of
pCDNA3 (Invitrogen) and pCDNA3/HIF-α such that all cells received the
same total quantity of DNA.

Cell Lysis and Immunoblotting.

Whole-cell protein extracts were prepared from tissue culture cells by
10-s homogenization in denaturing conditions as described
(31)
. Whole-cell protein extracts were prepared from
tumors by fine section of frozen tissue and 30-s homogenization in
denaturing conditions identical to tissue culture extracts. For Western
analysis, aliquots were separated by SDS-PAGE and transferred to
Immobilon-P membranes. CA IX was detected using the mouse monoclonal
antihuman CA IX antibody M75 (1:50) as described
(32)
.
Horseradish peroxidase-conjugated goat-antimouse immunoglobulin (DAKO;
1:2000) was applied for 1 h at room temperature. ECL Plus
(Amersham Pharmacia) was used for visualization.

Immunohistochemistry.

Formalin-fixed, paraffin-embedded tissue specimens collected by
standard surgical oncology procedures were obtained from the Pathology
Department, John Radcliffe Hospital (Oxford, United Kingdom).
Immunostaining of paraffin sections was performed after dewaxing and
rehydrating 4-μm sections. For CA IX detection, endogenous peroxidase
was blocked with 0.5% hydrogen peroxide in water for 30 min. To block,
10% normal human serum in TBS was applied for 15 min. M75 (see“
Immunoblotting”; 1:50) was applied for 30 min at room temperature.
Secondary polymer from Envision kit (DAKO) was applied for 30 min at
room temperature. For pimonidazole detection, sections were digested
with 0.01% Pronase (Sigma) in PBS for 30 min at 37°C. Endogenous
peroxidase was blocked with 0.1% hydrogen peroxide in water for 30
min. To block, Protein Block (DAKO) was applied for 5 min.
Anti-pimonidazole IgG1 antibody (Natural Pharmacia; 1:100) was applied
for 1 h at room temperature. Biotinylated rabbit antimouse
secondary (DAKO; 1:200) was applied for 1 h at room temperature.
ABC complex horseradish peroxidase conjugate (DAKO) was applied for
1 h at room temperature. Visualization of CA IX and pimonidazole
staining was by diaminobenzidine substrate. Slides were counterstained
with hematoxylin before mounting in Aquamount (BDH). Substitution of
primary antibody with PBS was used as a negative control for both
antibodies.

CA IX and pimonidazole were studied in semiserial tissue sections. The
percentage of tumor cells showing positive staining for CA IX or
pimonidazole and the extent of overlap between these regions within
each tissue section was assessed by light microscopy at low
magnification by three observers (C. C. W., P. H. W., and H. T.)
and a consensus was determined.

In Situ mRNA Hybridization.

Specific localization of VEGF mRNA was accomplished by in
situ hybridization using an antisense riboprobe. Briefly,
pBluescript (Stratagene) containing 517 consecutive complementary
nucleotides of the VEGF121 transcript (439
consecutive nucleotides of which are complementary to
VEGF165, VEGF189, and
VEGF206) was linearized with EcoRV for
16 h at 37°C. Labeled transcripts were synthesized using T7
(antisense) and SP6 (sense) polymerase in the presence of[
35S]UTP (>800 Ci/mmol; Amersham Pharmacia).
The methods for pretreatment, hybridization, washing, and dipping of
slides in Ilford K5 for autoradiography were as described for
formalin-fixed, paraffin-embedded tissue
(33)
. The
presence of hybridizable mRNA in tissue sections was established in
semiserial sections using an antisense β-actin probe. Hybridizations
using a sense probe were used to control for nonspecific signal.
Autoradiography was at 4°C (two exposures per section for VEGF
visualization at 10 and 18 days), before developing in Kodak D19 and
counterstaining by Giemsa’s method. Sections were examined under
conventional and reflected light-/dark-field conditions.

Pimonidazole Administration.

Patients with squamous or basal cell carcinomas of the skin and
patients with newly diagnosed transitional cell bladder carcinoma were
studied. Signed informed consent was obtained in all cases.
Pimonidazole hydrochloride was selected as the hypoxia marker because
of its high water solubility, chemical stability, efficient tumor
uptake, and low toxicity. Patients received 500
mg/m2 of pimonidazole hydrochloride,
1-[(2-hydroxy-3-piperidinyl)propyl]-2-nitroimidazole hydrochloride
(Hypoxyprobe) in 100 ml of normal saline i.v. over 20 min. This dose is
25% of the maximum tolerated dose
(34)
. Patients with
tumors in skin underwent incisional or Trucut biopsy under local
anesthetic 2–24 h after pimonidazole infusion. Patients with bladder
carcinoma underwent transurethral resection of the tumor under general
anesthetic 2–24 h after pimonidazole infusion. Tissue samples were
immediately placed in 10% neutral buffered formalin, protected from
light, and then processed into formalin blocks.

RESULTS

VHL-dependent Regulation of CA9 and
CA12 mRNAs by Hypoxia.

Expression of mRNAs encoding CA9 and CA12 was
analyzed by RPA. To confirm the previous report of down-regulation by
pVHL
(29)
, we first examined expression in the
VHL-defective RCC line RCC4 and a stable transfectant expressing a
human VHL cDNA (RCC4/VHL). In normoxic cells, both mRNAs were
down-regulated by pVHL. However, when cells were exposed in parallel to
normoxia or hypoxia (0.1% oxygen), induction by hypoxia was observed
in RCC4/VHL cells, whereas in RCC4 cells the high level of expression
in normoxia was unchanged by hypoxia (Fig. 1A)
⇓
. We also examined expression in other RCC lines that are
either defective (KRL140, SKRC28, UMRC2, and 786-0) or wild type
(Caki-1) for VHL, and in a stable transfectant of 786-0 re-expressing
wild-type pVHL (WT 8). Representative results from three cell lines are
illustrated in Fig. 1A⇓
. In the VHL-defective cells, both
CA9 and CA12 were constitutively expressed and
unresponsive to changes in oxygen tension. In the wild-type VHL cell
lines, both genes, when expressed, were induced by hypoxia.

Induction of CA9 and CA12
mRNA by hypoxia. Cells were exposed to either normoxia
(N; 20% O2) or hypoxia (H;
0.1% O2) for 16 h. CA9 and
CA12 mRNA was examined by RPA. A,
induction by hypoxia in renal carcinoma-derived cell lines is VHL
dependent. RCC4, SKRC28, and UMRC2 are VHL defective. RCC4/VHL is a
pVHL stable transfectant of RCC4. Caki-1 is VHL competent.
B, induction by hypoxia in nonrenal-derived cell lines
from indicated tissue type. C, comparison of induction
by hypoxia and DFO (each applied for 16 h) in A549 cells.
LC, signal from internal control assay for the
constitutively expressed U6 small nuclear RNA.

To examine regulation by hypoxia across a range of cell types, we
performed RPAs for CA9 and CA12 on mRNA samples
from normoxic and hypoxic cultures of 11 additional cell lines derived
from five different tissue types: bladder (RT112 and EJ-28), bone (U2
O-S), breast (HBL-100, MDA-MB-435S, MDA-MB-468, MDA-MB-231,
and T-47D), cervical (HeLa), and lung (A549 and NCI-H460). With the
exception of bladder cell lines RT112 and EJ-28, every cell type
expressed one or both CA isoforms, and in each case where expression
was observed, it was induced by hypoxia. The amplitude of induction by
hypoxia was particularly high for CA9; mRNA levels were at
or below the limit of detection in normoxia, yet strikingly induced by
hypoxia. Representative illustrations of one cell line from each tissue
type are depicted in Fig. 1B⇓
. Because many hypoxia-inducible
genes are up-regulated by treatment of cells with the iron chelator DFO
(35)
, we also tested the effect of DFO and found a similar
induction of both CA9 and CA12 mRNA (Fig. 1C)
⇓
.

CA9 Promoter Analysis.

To investigate the unusually tight regulation of CA9 mRNA by
hypoxia, we tested for oxygen-dependent function of the CA9
promoter. In the first set of experiments, we tested luciferase
reporter genes containing ∼0.5 kb of CA9 5′ flanking
sequences (−506 to +43) and a deletion to nucleotide −173 (−173 to+
43) in transiently transfected HeLa cells. Both constructs showed very
low levels of activity in normoxic cells but were induced strongly by
hypoxia (Fig. 2A)
⇓
. By contrast, a similar reporter linked to a minimal SV40
promoter showed no induction by hypoxia.

Functional analysis of human CA9
5′-flanking sequences in transient expression assays. Left
panel, schematic diagram of reporter genes; the indicated
CA9 wild-type and mutant sequences were inserted 5′ to a
promoterless luciferase reporter gene. Arrow, 5′
transcriptional initiation site. Underlined sequence,
CA9 putative HRE. Right panels, reporter
gene activities in transiently transfected cells. CA9
promoter sequences are indicated to the left of each
column. SV40, control minimal SV40
promoter. A, activities in normoxic and hypoxic HeLa
cells. B and C, activities in wild-type
CHO (C4.5) cells (columns 1) and HIF-1α-deficient CHO
(Ka13) cells (columns 2). A,
hypoxia-inducible activity of the CA9 promoter.
B, hypoxia-inducible activity of the CA9
promoter is ablated in Ka13 cells. Cotransfection of HIF-1α restores
induction by hypoxia in Ka13 cells and augments CA9
promoter activity in both wild-type and Ka13 cells. In comparison,
minimal effects are seen on the SV40 promoter. C, a
minimal CA9 promoter retains HIF-1α-dependent,
hypoxia-inducible activity. Two mutations within the putative HRE, MUT1
and MUT2, completely ablate hypoxia-inducible activity, whereas basal
transcription is preserved. Columns, mean luciferase
activities corrected for transfection efficiency from a typical
experiment performed in duplicate. Each duplicate experiment was
repeated two to six times. Numbers to the right are the
ratios of hypoxic to normoxic expression of the indicated reporter
construct. Transfected cells were incubated at 20% O2 for
8 h and then incubated at 20% O2 (normoxia) or 0.1%
O2 (hypoxia) for 16 h.

To test whether these responses were dependent on HIF-1, we performed
further transfections using a CHO mutant cell (Ka13) that is
functionally defective for the HIF-1α subunit and cannot form the
HIF-1 transcriptional complex
(36)
. In the CHO wild-type
parental subline C4.5, the −173 nucleotide promoter conferred 17-fold
transcriptional induction by hypoxia. In contrast, in the
HIF-1α-deficient Ka13 subline, this hypoxic induction was absent
(Fig. 2B)
⇓
. Cotransfection of human HIF-1α restored
hypoxia-inducible activity to the CA9 promoter in the Ka13
cells and increased normoxic activity in both C4.5 and Ka13 (Fig. 2B)
⇓
. In C4.5 and Ka13 cells at 0.1%
O2, luciferase expression was increased 1.6- and
17-fold, respectively, by cotransfection of human HIF-1α. Thus,
hypoxia-inducible activity of the CA9 promoter is completely
dependent on HIF-1 and strongly influenced by the level of HIF-lα.
Activity of the CA9 promoter in Ka13 cells could also be
restored by cotransfection of HIF-2α, although normoxic activity was
higher and fold induction by hypoxic stimulation was reduced (data not
shown).

Inspection of the CA9 5′ flanking sequences revealed a
consensus HRE beginning 3 bp 5′ to the transcriptional start site,
orientated on the antisense strand, reading 5′-TACGTGCA-3′ (Fig. 2
⇓
,
left). To test the importance of this site, we constructed a
CA9 minimal promoter containing this sequence (−36 to +14).
This minimal promoter retained hypoxia-inducible activity in C4.5 cells
but had no inducible activity in Ka13 cells (Fig. 2C)
⇓
.
Absolute levels of activity were lower in comparison to the −173
nucleotide promoter construct, being reduced ∼8 fold, indicating that
although sequences −173 to −36 amplified promoter activity,
responsiveness to hypoxia was conveyed by the minimal sequence
containing the HRE. To confirm the importance of this HRE, two
mutations were made within its core (antisense strand): a 3-bp
substitution from CGT → AAA (MUT1), and a single substitution of G→
T (MUT2; Fig. 2
⇓
, left). Both mutations completely
ablated hypoxia-inducible activity, although basal activity was
preserved or slightly increased for MUT1 (Fig. 2C)
⇓
.

Regulation of CA IX Protein by Oxygen.

As a first step toward understanding the significance of
hypoxia-inducible expression of CA9 mRNA, the effect of
hypoxia was examined on CA IX protein levels in whole-cell extracts.
Immunoblots of representative cells using anti-CA IX monoclonal
antibody M75 are illustrated in Fig. 3A⇓
. Striking induction of CA IX protein by hypoxia was
observed in multiple cell lines, whereas the VHL-defective RCC4 cells
showed constitutive up-regulation of CA IX protein. Thus, hypoxic
up-regulation of CA9 mRNA is clearly reflected at the
protein level. We next examined the response of CA IX to increasing
degrees of hypoxia (Fig. 3B)
⇓
. The level of CA IX hypoxic
induction after 16 h of exposure increased with decreasing oxygen
tensions from 5 to 0.1%. Because the original description of CA IX was
as an antigen induced by culture of cells at high density
(37)
, we also compared the effects of culture at high
density with those of hypoxia. In normoxic cultures of A549 cells, high
density clearly induced CA IX, although the effect was considerably
smaller than that of hypoxia (Fig. 3C)
⇓
.

CA IX Expression in Human Tumors.

We next sought to determine whether regulation of CA9 by
hypoxia in tissue culture cells was reflected in patterns of expression
within naturally occurring human tumors. To confirm the specificity of
M75 immunostaining in our laboratory, we first compared
immunohistochemical staining with CA IX immunoblot signals in pellets
of cultured cells. Pellets were prepared from normoxic cultures of RCC4
and RCC4/VHL cells and processed in parallel for whole-cell protein
extraction and immunohistochemistry. In keeping with the immunoblotting
results, immunostaining of these sections with M75 revealed strong
membrane expression of CA IX in RCC4 cells (Fig. 4A)
⇓
and no staining in normoxic RCC4/VHL cells (Fig. 4B)
⇓
. Then, we compared immunostaining and immunoblots of
tissue extracts from similar regions of tumor and normal tissue in four
sets of paired samples from surgical excisions of head and neck tumors.
By immunostaining, CA IX expression was low or absent in normal tissue
surrounding the tumors but was expressed at significant levels in each
tumor specimen. Results of immunoblot analysis correlated closely with
immunostaining, signals being very low or undetectable in each normal
tissue sample, and correlated with the extent of CA IX immunostaining
in tumor samples (data not shown).

Of particular interest to regulation by hypoxia is the relationship of
CA IX expression to zones of tumor necrosis. This was first examined in
a series of nine tumors of head and neck, breast, and ovary, each of
which showed well-defined zones of necrosis. Three tumors of each type
were analyzed. In each specimen, a predominantly or even exclusively
perinecrotic expression pattern was observed for CA IX. Representative
sections from each tumor type are illustrated in Fig. 4
⇓
. Expression was
localized to the cellular membrane. Tracing a line from the necrotic
center to adjacent viable cells revealed a gradient of CA IX
expression, with the highest levels observed in cells closest to or
within necrotic regions (Fig. 4, C–F)
⇓
.

Because pVHL inactivation leads to loss of CA9 regulation by
oxygen in cultured cells and is common in clear cell renal carcinoma
but not other renal tumors, we next compared expression patterns in a
second series of 35 clear cell renal tumors and eight papillary renal
tumors. Representative sections from a clear cell and a papillary tumor
are illustrated in Fig. 4, G and H⇓
. Expression
patterns were markedly different. In 33 of 35 clear cell tumors, (both
sporadic and derived from VHL syndrome patients), CA IX was expressed
throughout tumor tissue; strong membrane staining was observed in tumor
cells, regardless of proximity to necrosis or vessels (G).
In contrast, in papillary renal tumors CA IX immunostaining was much
less evident but was observed in tumors containing areas of necrosis,
where, as with the nonrenal tumors, staining was strikingly focal and
perinecrotic (four of eight papillary tumors contained necrosis, and
all four showed focal CA IX positivity; Fig. 4H⇓
). Thus, the
tight regulation of CA9 expression by oxygen observed in
cell culture appeared to be reflected in strikingly focal patterns of
expression around areas of necrosis.

Relationship of CA IX Expression with an Endogenous and an
Administered Hypoxia Marker in Human Tumors.

To compare CA IX expression with potential markers of tumor hypoxia, we
examined expression of VEGF mRNA and activation of the bioreductive
hypoxia marker pimonidazole in relationship to CA IX staining. Serial
sections of a subset of our first series of tumors were analyzed for
VEGF mRNA expression by in situ hybridization, and CA IX
expression was analyzed by immunostaining. Representative views from an
ovarian and head and neck tumor sample are illustrated in Fig. 5
⇓
. VEGF mRNA was expressed at varying levels throughout tumor tissue but
was increased greatly in regions adjacent to necrosis. CA IX
immunostaining showed strong overlap but was more tightly limited to
perinecrotic regions.

Comparison of expression patterns for CA IX and VEGF in
tumor biopsies. Immunohistochemical detection of CA IX using anti-CA IX
monoclonal antibody M75 and in situ mRNA analysis of
VEGF is shown. A, C, and E, ovarian
adenocarcinoma. B, D, and F, head and
neck carcinoma. A and B, CA IX
immunostaining. C and D, dark-field views
of in situ hybridization for VEGF mRNA on sections
serial to the CA IX-immunostained sections. E and
F, bright-field views of VEGF in situ.
Arrows within necrotic areas (n) in bright-field
views point toward the boundary with viable tumor cells.
s, stroma; t, regions of viable tumor
cells. All panels, ×100.

For comparison of pimonidazole staining with CA IX expression, a series
of 14 transitional cell bladder carcinomas and 6 squamous or basal cell
skin carcinomas derived from patients who had received pimonidazole
prior to surgical excision of tumor tissue was analyzed. Representative
views of pimonidazole- and CA IX-stained sections are illustrated in
Fig. 6
⇓
, and assessment of pimonidazole and CA IX staining with corresponding
overlap for each tumor biopsy are indicated in Table 1
⇓
. In most tumors (16 of 20), pimonidazole staining was more extensive
across tumor sections than CA IX staining, being primarily banded
around necrotic areas (Fig. 6, C and E)
⇓
and the
periphery of papillary structures in bladder carcinomas (Fig. 6A)
⇓
. Although less extensive, the large majority of CA IX
immunostaining localized within regions of pimonidazole adduct
formation and was also associated with necrosis (Fig. 6, D and F)
⇓
or the periphery of papillary structures in bladder
carcinomas (Fig. 6B)
⇓
. Some regions containing CA IX were
observed that were slightly farther removed from necrosis than regions
staining positive for pimonidazole (Fig. 6, C and D)
⇓
. In 4 of 20 cases, CA IX staining was more extensive than
pimonidazole staining. In these cases, in addition to the
characteristic perinecrotic and peripheral papillary expression, a
proportion of CA IX expression was not obviously associated with such
regions in the plane of the section. Nevertheless, within these four
tumors the pimonidazole-positive regions were consistently localized
within regions of CA IX positivity, again demonstrating the overlap
between these markers. Despite the relationship between pimonidazole
and CA IX at the microscopic level in all tumors, we did not observe an
overall correlation between the percentage of tumor stained for
pimonidazole and CA IX (Table 1)
⇓
.

DISCUSSION

In this work, we have demonstrated that the tumor-associated CAs
CA9 and CA12 are strongly inducible by hypoxia in
a broad range of tumor cells. Our findings also explain up-regulation
of these CA isoforms in VHL-defective renal tumors, indicating that
they are expressed constitutively at a high level in VHL-defective
cells as a consequence of constitutive activation of HIF. The work
therefore extends the range of HIF target genes to a new class of
molecule that may have important implications for understanding the
consequences of microenvironmental tumor hypoxia, as well as the
tumor-promoting effects of VHL inactivation. The regulation of
CA9 was particularly tightly controlled by oxygen, and we
analyzed the hypoxia- inducible response of this gene in detail.

Studies of the CA9 promoter demonstrated that sequences
close to the transcriptional initiation site were sufficient to convey
a hypoxia-inducible response, that this activity was mediated by HIF,
and that it was dependent on a consensus HRE lying adjacent to the
initiation site. The CA9 promoter contains neither a TATA
box nor a consensus initiator sequence at the cap site
(38)
. The association of this unusual anatomy with tight
regulation by hypoxia is therefore of interest and suggests that it may
be informative to pursue the mechanism by which HIF interacts with the
basal transcriptional machinery operating on this gene. Furthermore,
irrespective of the mechanism, the strong inducibility conveyed by the
minimal CA9 promoter is unusual and may itself be of
utility, for instance in the refinement of gene therapy vectors seeking
to target therapeutic gene expression to hypoxic regions of tumors
(39, 40)
.

Our findings also raise a number of issues relevant to recently
published analyses of the CA9 promoter that did not examine
the effect of hypoxia: (a) they provide an explanation for
the remarkably low levels of CA9 promoter activity recently
reported under standard culture conditions
(41)
, because
promoter activity is so strongly dependent on hypoxia; (b)
they are consistent with the positive activity demonstrated for
sequences −173 to +31
(41)
and show that the
transcriptional effects mediated by these sequences interact with the
HRE in the minimal promoter to amplify the response to hypoxia;
(c) they are consistent with the absence of a DNase I
footprint in the region of the HRE
(41)
, because even in
hypoxia it has been shown that HIF-1 binding characteristics are such
that an in vitro footprint is not demonstrated
(42)
; (d) they provide a potential explanation
for the repressive effects of p53 expression on the activity of the
CA9 promoter in some cells
(43)
, because it has
been suggested that p53 can interact with the regulation of HIF-1α
stability so as to reduce activity of the HIF/HRE complex
(15, 16)
.

In tissue culture, CA9 demonstrated a very marked difference
between constitutive expression in VHL-defective RCC cells and strong
induction by hypoxia in cells known or presumed to be VHL competent.
This provided an opportunity to determine the extent to which these
contrasting patterns of regulation in culture were reflected in
patterns of expression within native tumors. In our series of renal
tumors, we found a striking contrast between generalized expression in
clear cell carcinomas, which are usually defective in VHL, and focal
perinecrotic expression in papillary renal tumors, which are usually
wild type for VHL. Notwithstanding the absence of direct ascertainment
of VHL genotype in all of the tumors analyzed, this strongly suggests
that effects of VHL status on HIF-dependent, hypoxia-inducible gene
expression are reflected in patterns of expression within native
tumors. Up-regulation by constitutively active HIF therefore provides
an explanation for the utility of CA9 as a marker for clear
cell carcinoma. The pattern of diffuse expression in clear cell
carcinoma is in agreement with findings of a previous analysis of CA IX
expression in which the authors focused on high levels of expression in
clear cell carcinoma versus absent expression in a variety
of benign lesions and postulated that CA IX expression might be useful
as a marker of malignant change
(25)
. That study also
noted focal expression in papillary renal carcinoma, although the
authors did not comment on the relation to necrosis. In our studies, we
found that the striking localization of focal CA IX expression to zones
of necrosis is not just observed in papillary renal carcinoma but also
in several series of nonrenal tumors. The pattern is similar to that
first described for VEGF mRNA
(5)
, and we compared
directly the pattern of CA IX immunostaining with that of in
situ mRNA hybridization for VEGF in several types of tumors. In
this work, we used in situ mRNA hybridization for VEGF to
localize the site of production, because, in contrast with CA IX, some
isoforms of VEGF are secreted. Patterns of expression for CA IX and
VEGF mRNA were clearly concordant. However, CA IX expression was more
strikingly delimited, being essentially limited to regions surrounding
zones of necrosis.

The concordance of hypoxia-inducible versus constitutive
patterns of expression in tissue culture with focal perinecrotic
versus diffuse patterns of expression in tumors strongly
supports the view that the focal perinecrotic pattern of expression is
driven by microenvironmental hypoxia. Furthermore, the particularly
tight regulation of CA9 by hypoxia suggested that it might
be useful as a hypoxia marker. It was, therefore, of interest to
compare the pattern of CA IX immunostaining with staining for the
hypoxia marker pimonidazole
(44,
45,
46)
. Our analysis
demonstrated clear overlap of the staining patterns, supporting
expression of CA IX in hypoxic regions. Previous studies have compared
the distribution of immunodetectable pimonidazole adducts with VEGF
immunostaining. One study concluded that pimonidazole and VEGF
displayed the same pattern of staining on adjacent sections during the
angiogenesis associated with a model of liver fibrogenesis
(47)
, whereas an earlier study emphasized the
discrepancies between pimonidazole and VEGF staining, although regions
of overlap were demonstrated
(48)
. Among the explanations
considered for the differences between the distribution of VEGF
staining and pimonidazole adducts were regulation of VEGF by nonhypoxic
stimuli and diffusion of VEGF from hypoxic sites of production. For
CA9, basal expression in normoxic cells was lower than we
have observed for VEGF, induction by hypoxia was more striking, and the
protein was not secreted. Despite this, we also observed differences in
pimonidazole and CA IX staining. The substantial regions of overlap
presumably reflect regions where tumor hypoxia was of sufficient
duration and severity to activate both markers. Regions of nonoverlap
could reflect the operation of additional positive or negative
influences on expression or activation or different time frames of
induction or activation. For instance, pimonidazole adducts are formed
over a relatively short period of time and are then long-lived
(45, 46)
, whereas we have found that CA IX is a stable
protein that, in tissue culture, accumulated over a long period of
hypoxia (data not shown). Thus, CA IX induction might only be expected
in regions of relatively chronic tumor hypoxia and would reflect a
different hypoxic time frame from pimonidazole activation. Correlation
of focal CA IX expression with direct measurements of tumor oxygenation
and with clinical parameters of outcome will be of interest.

The demonstration that an extracellular CA is up-regulated by
microenvironmental tumor hypoxia has potentially important implications
for understanding the regulation of tumor pH and the response to
hypoxia. It has been widely held that lactate production by
glycolysis is a major cause of the acidic extracellular pH of
tumors
(49)
, and indeed glycolytic enzymes are induced by
hypoxia
(11)
, as is lactate production
(50)
.
However, tumors grown from mutant cells with glycolytic defects show a
similar extracellular acidosis in the absence of lactate accumulation
(51, 52)
, indicating that other mechanisms must be
involved. Recently, it has been proposed that extracellular CAs could
convert CO2 diffusing from oxygenated areas to
carbonic acid and promote the generation of bicarbonate and hydrogen
ions
(29, 52)
. Bicarbonate might then be exchanged for
intracellular chloride, providing a mechanism for maintaining the
characteristic extracellular acidosis and intracellular alkalosis that
is postulated to promote tumor growth
(53)
. Thus, it is
likely that the hypoxia-inducible behavior of tumor-associated CAs
could exert important biological effects through an influence on
microenvironmental pH. This could have therapeutic implications because
CA inhibitors have been shown to inhibit the invasion of renal cell
carcinoma lines in model culture systems
(54)
and have
synergistic effects with other chemotherapeutic agents in animal models
(55)
. The potential for strong induction by hypoxia will
now need to be considered in assessing the diagnostic and therapeutic
implications of tumor-associated extracellular CAs.

Acknowledgments

Footnotes

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

↵1 This work was supported by the Wellcome Trust
and the Imperial Cancer Research Fund.